WO2012012612A2 - Ligature tétrazine-trans-cyclooctène pour la construction rapide de sondes marquées par un radionucléide - Google Patents

Ligature tétrazine-trans-cyclooctène pour la construction rapide de sondes marquées par un radionucléide Download PDF

Info

Publication number
WO2012012612A2
WO2012012612A2 PCT/US2011/044814 US2011044814W WO2012012612A2 WO 2012012612 A2 WO2012012612 A2 WO 2012012612A2 US 2011044814 W US2011044814 W US 2011044814W WO 2012012612 A2 WO2012012612 A2 WO 2012012612A2
Authority
WO
WIPO (PCT)
Prior art keywords
diels
tetrazine
alder adduct
residue
mmol
Prior art date
Application number
PCT/US2011/044814
Other languages
English (en)
Other versions
WO2012012612A3 (fr
Inventor
Joseph M. Fox
Matthew Hassink
Melissa Blackman
Zibo Li
Peter S. Conti
Original Assignee
University Of Delaware
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Delaware filed Critical University Of Delaware
Priority to US13/811,568 priority Critical patent/US10434197B2/en
Priority to CN201180041210.XA priority patent/CN103221398B/zh
Priority to EP11810394.4A priority patent/EP2595967B1/fr
Publication of WO2012012612A2 publication Critical patent/WO2012012612A2/fr
Publication of WO2012012612A3 publication Critical patent/WO2012012612A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0497—Organic compounds conjugates with a carrier being an organic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D257/08—Six-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • Positron emission tomography is a non-invasive imaging modality that utilizes positron-emitting radionuclides (C-ll, N-13, 0-15 and F-18).
  • F-18 PET has a number of attributes that make it clinically attractive, including 100% positron efficiency, a very high specific radioactivity, and a short half-life of ⁇ 110 min.
  • the short half-life of F-18 and the poor nucleophilicity of fluoride render it difficult to incorporate F-18 in complex molecules.
  • radiochemistry is a major limiting factor for the field of PET.
  • challenges exist for improving F-18 incorporation with respect to reaction rates, efficiency, and selectivity.
  • tetrazine-trans-cyclooctene ligation OTTCO ligation' as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis
  • tra/is-Cyclooctene derivatives are readily prepared from c/ ' s- cyclooctenes using a photochemical flow-reaction that the inventors developed (M.
  • 3,6-diaryl-s-tetrazines offer an excellent combination of fast reactivity and stability for both the conjugate and starting material.
  • 3,6 - di(2-pyridyl)-s-tetrazines have been shown to display excellent characteristics.
  • the reaction between frans-cyclooctene and la proceeds with a rapid rate (k 2 ⁇ 2000 M 1 s "1 in 9: 1 MeOH :water), and is successful in cell media and cell lysate.
  • 3,6 -Di(2-pyridyl)- s-tetrazines can easily be functionalized as their amido derivatives ( lb), which display excellent st
  • the TTCO-ligation offers opportunities for the rapid conjugation of radionuclides to biomolecules, both of which are often available only at low concentration.
  • the invention provides a Diels-Alder adduct of a trans-cyclooctene with a tetrazine, wherein the adduct bears a substituent labeled with a radionuclide.
  • the invention provides a method of producing a PET or other image of an organ in an animal or human.
  • the method includes forming in the animal or human a Diels-Alder adduct of a trans-cyclooctene with a tetrazine, wherein the adduct bears a substituent labeled with a radionuclide.
  • the invention provides the compound according to structure 5
  • the invention provides the compound according to structure 9
  • the invention provides the compound according to structure 17
  • the invention provides the compound according to structure 13
  • the invention provides the compound according to structure 24 vides the compound according to structure 25
  • the invention provides the compound according to structure 26 provides the compound according to structure 27 BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 shows cell binding affinity studies of c(RGDyK) ( 12) and 19 F-15.
  • Figure 2 is a schematic representation of an automated synthesis module for performing fluorination ( ⁇ 200 mCi).
  • Figure 3A shows tumor and major organ uptake of 18 F-41.
  • the invention provides a number of ways to use TTCO-ligation for labeling, as will now be described. Labeling with 18 F will be described first, followed by a description of labeling with other radionuclides.
  • the inventors focused on the development of direct methods for 18 F-incorporation via reactions with fluoride ion, with an initial focus on the synthesis of 18 F-labeled tetrazines (Scheme 1). Attempts to convert nitrotetrazine derivatives 2a and 2b into 18 F-labeled substitution products 3 using 18 F-fluoride/kryptofix or 18 F-TBAF gave decomposition products and only traces of radiolabeled products. The inventors also combined mesylate 4 with fluoride, and in the most successful experiment ( 18 F-TBAF at 85°C for 15 min) 18 F-labeled product 5 was obtained in ⁇ 1% labeling yield.
  • Nosylate 8 reacted efficiently with TBAF to provide 19 F-9 in high yield.
  • the notation 19 F- as part of a structure name indicates that the compound has not been radiolabeled, and 18 F- indicates labeling .
  • the invention provides analogs of 18 F-9 in which the spacer group between 0 and 18 F is an alkylene group.
  • the alkylene group may be a C2 alkylene group (as in 18 F-9), or it may be any alkylene group from C3 to C20, more typically from C3 to CIO, and most typically from C3 to C5.
  • an analog of 18 F-9 uses an 18 F-CH 2 -phenyl moiety in place of the 18 F-CH 2 - CH 2 moiety of 18 F-9.
  • Such a compound may be prepared from the corresponding bromide, chloride, iodide, sulfonate, etc. by nucleophilic displacement with 18 F fluoride.
  • the inventors also used a photochemical flow reactor (M. Royzen, G. Yap, and J. Fox, J. Am. Chem. Soc. 2008, 130 ( 12), 3760) to synthesize 16, an analog of 7 with a functionalizable group.
  • reaction between la and 16 was even faster, in fact too fast to measure by UV-vis spectroscopy.
  • 3,6-di(2-pyridyl)-s-tetrazine e.g ., lb
  • known methods for example as described by M . L. Blackman, M. Royzen and J. M. Fox, J. Am. Chem. Soc. 2008, 130, 13518-13519 and R. Rossin, P. R. Verkerk, S. M. v. d . Bosch, R. C. M. Vulders, I. Verel, J . Lub and M . S. Robillard, Angew. Chem. Int. Ed.
  • the Diels-Alder conjugate 10 was found to be stable in water, and the benign isomerization to 11 was the only side reaction. Thus, 19 F-10 was the only product detected by : H NMR analysis immediately after the conjugation. In CD 3 CN/H 2 0, the rearrangement of 19 F-10 to 19 F-11 proceeded to 11% conversion after 4 hours, and >95% conversion after 48 hours. The stability of the radiolabeled conjugation product was monitored in PBS buffer and serum media for 4 hours, and no degradation products of 18 F-10 were observed.
  • the inventors report the construction of a 18 F labeled PET probe for imaging (such as cancer imaging) based on the tetrazine-trans-cyclooctene ligation.
  • the reaction speed for labeling tetrazine-RGD peptide 14 with 18 F-9 was then investigated .
  • 18 F-9 was synthesized from nosylate 8.
  • the conjugation of 18 F-9 and tetrazine-RGD was performed at room temperature for 5 min.
  • the conjugation was efficient and high yielding : starting with only 30 pg (78 ⁇ ) of tetrazine-RGD conjugate 14 and 2 mCi (5 ⁇ ) of 18 F-rrans-cyclooctene, the labeling yield was 90% after 5 min by HPLC analysis.
  • the stability of 18 F-tetrazine-RGD conjugate 15 was evaluated in PBS in the presence 5% EtOH. The stability was excellent: >95% of the tracer remained after incubation for 6 h as judged by radio-HPLC analysis.
  • Static microPET scans were performed for 18 F-tetrazine-RGD conjugate 15, via injection into athymic female nude mice bearing an U87MG tumor. High tumor accumulation was observed as early as 30 min time point. The tumor uptake was 4.6 ⁇ 0.2, 4.4 ⁇ 0.6, 4.2 ⁇ 0.6, and 2.7 ⁇ 0.5 %ID/g at 0.5, 1, 2, 4 h post injection, respectively. 18 F-15 was cleared through both liver and kidney. Up to 4 h post injection, there was still a fair amount of activity accumulated in the abdomen, which may be a result of the relative hydrophobicity of this system.
  • Blocking experiments were also performed by co-injecting 10 mg/kg of c(RGDyK) along with 18 F-15.
  • the tracer uptake in U87MG tumor dropped to 1.4 ⁇ 0.2, 1.0 ⁇ 0.3, 0.6 ⁇ 0.2, and 0.4 ⁇ 0.1 %ID/g at 0.5, 1, 2, 4 h post injection respectively.
  • the successful blocking confirmed the integrin ⁇ ⁇ ⁇ 3 specific binding of the tracer 18 F-15.
  • conjugation of 18 with 9 to give conjugate 19 is extremely rapid and extremely efficient, and conjugate 19 oxidized spontaneously in solution to form the aromatized compound 20.
  • the inventors have synthesized the diaminotetrazine 21 and derivative 22, which can be expected to show rapid conjugation with trans-cyclooctenes such as 9 while affording robust in vivo stability.
  • the invention provides efficient methods for conjugating tetrazine and irans-cyclooctenes to proteins.
  • the amine-reactive NHS- ester 13 can be readily converted into the thiol-reactive maleimide derivative 24.
  • the reactive functionalities of these molecules may be used to conjugate tetrazines to lysine and cysteine residues of proteins, for example a VEGF protein.
  • NHS-ester 13 can be prepared from 5-oxo-5-(6-(6-(pyridin-2-yl)-l,2,4,5-tetrazin-3-yl)pyridin-3- ylamino)pentanoic acid, in turn prepared by the method of R. Rossin, P. R.
  • the inventors have conjugated maleimides 26 and 27 to a protein (thioredoxin) using standard methods known in the art, and the conjugates have been shown to undergo rapid tetrazine ligation in high yield .
  • Other proteins may be similarly conjugated according to the invention.
  • Compound 33 the conjugate of 27 with thioredoxin, was rapidly adducted with 3,6-di(2-pyridyl)-s-tetrazine ( la) as shown below, where Trx represents a thioredoxin residue.
  • VEGF proteins may be labeled with 18 F via modification of the lysines of wild -type VEGF 12 i protein with amine reactive derivatives (e.g., 24 or 25).
  • the resulting conjugation products (29 and 30, respectively) may be combined with irsns-cyclooctene 18 F-17 and tetrazine 18 F-5 to provide radiolabeled conjugates 18 F-31 and 18 F-32, respectively.
  • Labeling of biomolecules with radionuclides using tetrazine -frans-cyclooctene ligation is not limited to the specific frans-cyclooctenes and tetrazines discussed above; any radiolabeled tetrazine or rrans-cyclooctene may be used.
  • the invention encompasses labeling of biomolecules with radionuclides other than F. Suitable further examples of compounds according to the invention, all of which may be used to label
  • I labeling may for example be suitable for producing PET probes, while 125 I and 131 I may be suitable for preparing SPECT probes.
  • IR liquid, CHCI 3 , cm “1 ) 3456, 3011, 2975, 2936, 2861, 1650, 1447, 1392, 1252, 1100, 1049, 988, 875.
  • the silica gel was filtered and the filtrate was placed into a separatory funnel.
  • the organic layer was separated, and the ammonium hydroxide layer was extracted three times with methylene chloride.
  • the organic layers were combined and twice washed with water.
  • the organic layers were dried with MgS0 4 , filtered, and purified by column chromatography with 5% ether to 30% ether in hexanes.
  • IR liquid, CHCI 3 , cm “1 ) 3449, 3012, 2935, 2861, 1647, 1445, 1353, 1198, 1096, 1050, 993, 968, 797.
  • IR liquid, CHCI 3 , cm “1 ) 3105, 3010, 2935, 1609, 1536, 1351, 1187, 1097, 932, 857, 776, 616.
  • the purification of the crude product was carried out on a analytical reversed - phase high performance liquid chromatography (HPLC) system equipped with a dual UV absorbance detector (Waters 2487) using a Phenomenex C18 RP ( 150 x 4.6 mm 5 micron).
  • the flow was 1 mL/min, with the mobile phase starting from 95% solvent A (0.1% TFA in water) and 5% solvent B (0.1% TFA in acetonitrile) (0-2 min), followed by a gradient mobile phase to 5% solvent A and 95% solvent B at 17 min, which was then kept at 95% B until 22 min.
  • the radioactivity was detected by a model of Ludlum 2200 single-channel radiation detector.
  • a semi-preparative C18 reverse, phase column (Phenomenex C18) was used in separations with a 4 mL/min flow rate under gradient conditions.
  • the 18 0 is in the form of water with an isotopic purity of greater than 95%.
  • the operation of the cyclotron and target functions was automatically controlled by the cyclotron computer system.
  • the target was loaded with the required amount of [ 18 0]-water, and bombarded for the appropriate time with a suitable beam current.
  • the target was then unloaded to a collection vial located in the dose calibrator; where the amount of fluoride was measured.
  • the fluoride solution was then transferred to the chemistry operation.
  • the fluorination ( ⁇ 200 mCi) was performed on an automated synthesis module as shown in Figure 2.
  • two-way valves V1-V6 were used to control the solvent and reagent containing reservoirs 1-6.
  • Reservoirs 3-6 are connected with a nitrogen or argon gas line.
  • Reservoir 1 is connected with reactor through several control valves.
  • the reactor is connected with vacuum pump, gas line, and the injection port of the HPLC system.
  • the solutions of potassium carbonate and Kryptofix K2.2.2 (or TBAB and MeCN) were loaded into Reservoirs 1 and 2, respectively.
  • Reservoirs 3, 4, 5, and 6 were filled with precursor solution and other chemicals/solutions as needed.
  • Reservoir 2 was added into the reactor and the whole mixture was dried at 95°C in combination with nitrogen flow and vacuum .
  • the precursor solution from Reservoir 3 was added to the dried 18 F ion and heated at the desired temperature.
  • the reaction mixture will be sampled out for analysis or loaded on HPLC for purification.
  • Tetrazine 4 was prepared by mesylation of (MsCI, Et 3 N, CH 2 CI 2 ) of 3-(4-hydroxymethyl)-phenyl-s-tetrazine, which had been prepared from 4-hydroxymethylbenzonitrile (formamidine acetate, S 8 , hydrazine hydrate, then NaN0 2 /HOAc), according to the method of S. A. Lang Jr., B. D. Johnson, E. J. Cohen, J. Heterocycl. Chem. 1975, 12, 1143. Compound 4 was dissolved in MeCN and then allowed to react with 18 F-TBAF at 85°C for 15 min. The reaction mixture was then analyzed by HPLC.
  • Fluoride was dried as described above.
  • Precursor 8 was dissolved in MeCN and added to the azeotropically dried fluoride from Reservoir 3.
  • the crude mixture was heated at desired temperature and then analyzed by HPLC.
  • the optimized labeling conditions are described in entry 3 in table 2.
  • the crude reaction mixture was loaded onto the semi-prep HPLC for separation.
  • the purified sample was injected to the analytical HPLC.
  • 18 F-9 was eluted off at 17.4 min on HPLC, which correlates with the retention time of the standard compound . Under the optimized conditions, the radiochemical purity of 18 F-9 was more than 98%.
  • HPLC-purified 18 F-9 was mixed with 3,6-Di(2-pyridyl)-s-tetrazine ( la) under the conditions described in Table 2. Immediately after mixing, the crude reaction mixture was analyzed by HPLC. The HPLC injection was made within 10 seconds of mixing. 18 F- 10 was eluted off at 11.8 min on HPLC, which correlates with the retention time of the standard compound . Small amounts of isomers 18 F-11 were also observed on the HPLC radio trace. The retention time of peaks attributable to the isomers of 18 F-11 were 12.7 and 13.3 min, which correlates with the retention times for the 19 F standards.
  • [ 18 F] Fluoride was prepared by the 18 0(p,n) 18 F nuclear reaction, and it was then adsorbed onto an anion exchange resin cartridge.
  • Kryptofix 222/K 2 C0 3 solution 1 mL 9 : 1 acetonitrile/water, 15 mg Kryptofix 222, 3 mg K 2 C0 3 ) was used to elute the cartridge, and the resulting mixture was dried in a glass reactor.
  • 18 F-9 was prepared according to reported procedures and purified via semipreparative HPLC. Labeling of tetrazine-RGD conjugate 14 with 18 F-9 was performed in DMSO/EtOH (1 : 3). The resulting mixture was diluted with water and purified by semi -preparative HPLC. The final product 18 F-15 was concentrated and formulated in saline (0.9%, 500 ⁇ ) for in vivo studies.
  • U87MG human glioblastoma cells were grown in Dulbecco's medium (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 pg/mL streptomycin (Invitrogen Co.). Animal procedures were performed according to a protocol approved by the Stanford University Institutional Animal Care and Use
  • ROIs regions of interest
  • the radioactivity concentration (accumulation) within a tumor was obtained from the mean value within the multiple ROIs and then converted to %ID/g.
  • mice bearing U87MG tumors on the front left flank were scanned after co-injection with 18 F-15 and c(RGDyK) (10 mg/kg).
  • the vial was swept with n itrogen, and anhydrous DMF ( 1.5 mL) was added via syringe.
  • the reaction mixture was allowed to stir for 20 h at room temperature.
  • the mixture was then diluted by CH 2 CI 2 (5 mL), centrifuged, and the supernatant was decanted.
  • the purple solid was subjected to three further cycles of suspension in CH 2 CI 2 (5 mL), centrifugation, and decantation to provide the 130 mg (59%) of the title compound as a purple solid ( 130 mg, 59%) .
  • a dry 3 mL vial was charged with 13 (5.2 mg, 0.011 mmol) and 12 (3.4 mg, 0.0055 mmol).
  • the vial was capped by a screw cap with a Teflon septum, and the vial was swept with nitrogen.
  • a solution of diisopropylethylamine ( 1.4 mg, 0.011 mmol) in DMF (30 pL) was added via syringe, followed by the addition of DMF (0.57 mL).
  • the reaction mixture was allowed to stir for 18 h at room temperature.
  • the mixture was then diluted by methanol (1 mL), centrifuged, and the supernatant was decanted .
  • the purple solid was subjected to two further cycles of suspension in methanol ( 1 mL), centrifugation, and decantation to provide 14 as a purple solid (5.1 mg, 96%) .
  • the purity was judged to be 95% by HPLC analysis (Shimadzu C18 column, 4.6 mm x 50 mm, 5 micron). HPLC analysis was performed with eluents that contained 0.1% trifluoroacetic acid, eluting at 1 mL/min, initially with 10% acetonitrile/water for 5 min, followed by a gradient of 10%-20% acetonitrile/water for 5 min, and final elution with 20% acetonitrile/water.
  • HPLC analysis was performed with eluents that contained 0.1% trifluoroacetic acid, eluting at 1 mL/min, initially with 5% acetonitrile/water for 2 min, followed by a gradient of 5%-95% acetonitrile/water for 22 min, and final elution with 95%
  • Tetrazine-R conjugate 18 was prepared according to the following sequence.
  • a dry round-bottom flask was charged with 37 (0.804 g, 2.00 mmol), CH 3 CN (15 mL) and 64% hydrazine hydrate (0.097 mL, 2.00 mmol).
  • the flask was fitted with a reflux condenser, and the mixture was heated to 50 °C for 1 h behind a blast shield.
  • K 2 C0 3 (0.553 g, 4.00 mmol) was added, and the reaction mixture was allowed to reflux and stir for 24 h.
  • 64% hydrazine hydrate (0.291 mL, 6.00 mmol) was added to the mixture, which was allowed to reflux for an additional hour.
  • the reaction mixture was cooled to room temperature.
  • the resulting orange precipitate was isolated by filtration, washed with cold H 2 0, and dried under vacuum.
  • the crude residue was diluted with glacial acetic acid (4.0 mL) at 0°C, and a solution of NaN0 2 (0.690 g, 10.0 mmol) in H 2 0 (1.1 mL) was added dropwise to the solution.
  • the reaction mixture was diluted with CH 2 CI 2 (100 mL) and washed repeatedly with saturated NaHC0 3 .
  • the organic solution was dried over MgS0 4 , filtered, and concentrated in vacuo.
  • a resealable glass vial was flushed with N 2 and charged with 36 (0.010 g, 0.0320 mmol), glutaric anhydride (0.018 g, 0.158 mmol) and anhydrous THF (0.3 mL). The vial was capped, and the mixture was stirred at 80 °C for 1 h. The reaction mixture was cooled to room temperature, triturated with CH 2 CI 2 and hexanes and dried under vacuum to give 0.010 g (0.023 mmol, 72%) of 35 as a pink solid, mp 246-248°C.
  • a dry 3 mL vial was sequentially charged with 5-oxo-5-((3-(6-(4- (trifluoromethyl)phenyl)-l,2,4,5-tetrazin-3-yl)phenyl)amino)pentanoic acid (170 mg, 0.46 mmol), /V-hydroxysuccinimide (86 mg, 0.20 mmol) and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (77 mg, 0.40 mmol).
  • the vial was capped by a screw cap with a Teflon septum.
  • the vial was swept with nitrogen, and anhydrous DMF (1.5 mL) was added via syringe.
  • the reaction mixture was allowed to stir for 34 h at room temperature.
  • the mixture was then diluted by CH 2 CI 2 (10 mL), centrifuged, and the supernatant was decanted.
  • the purple solid was subjected to three further cycles of suspension in CH 2 CI 2 (10 mL), centrifugation, and decantation to provide the 79 mg (75%) of the title compound as a purple solid (79 mg, 75%). !
  • a tetrazine conjugate 41 was prepared from c(RGDyC) (39) according to the following sequence.
  • tetrazine-maleimide 200 pg, 0.41 pmol
  • 100 pL dimethyl sulfoxide (DMSO) and 39 (200 pg, 0.33 pmol) (Peptides International of Louisville, KY) in 500 pL phosphate buffer (50 mM, pH 6.5-7.0) were mixed together at room temperature. After the mixture was stirred at room temperature for 5 h, the conjugate was purified by semipreparative HPLC. The collected fractions were combined and lyophilized to afford the final product as a white powder. Compound 40 was obtained in 85% yield.
  • tetrazine-maleimide 24 200 pg, 0.41 pmol in 100 pL DMSO and VEGF (100 pg, 5.5 nmol) in 500 pL phosphate buffer (50 mM, pH 6.5-7.0) were mixed together at room temperature. After the mixture was stirred at room temperature for 5 h, the conjugate was purified by size exclusion PD-10 column and concentrated by Centricon filter (Millipore, Bedford, MA), and the final concentration was determined based on UV absorbance at 280 nm using unconjugated VEGF of known concentrations as standard. The final concentration was adjusted to 50 pg/mL for use.
  • 19 F-9 was synthesized according to Li Z, Cai H, Hassink M, Blackman ML, Brown RC, Conti PS, et al. Tetrazine-trans-cyclooctene ligation for the rapid construction of 18F labeled probes.
  • 19 F-41 was obtained in 92% yield with 15.5 min retention time on analytical HPLC.
  • MALDI-TOF-MS was m/z 1226.4 for [MH] + (C57H73FN15O13S, calculated molecular weight 122
  • 18 F-9 was synthesized according to Li Z, Cai H, Hassink M, Blackman ML, Brown RC, Conti PS, et al. Tetrazine-trans-cyclooctene ligation for the rapid construction of 18F labeled probes. Chem Commun (Camb). 2010;46:8043-8045. 18 F-9 (148 MBq, 4 mCi) in about 50 pL ethanol was added to 40 (10 pg) in 50 pL DMSO followed by shaking for 5 min. The conjugate was purified by semipreparative HPLC. The collected fractions were combined and the solvent was removed by rotary evaporation under reduced pressure. 18 F-41 was reconstituted in 1 mL PBS and passed through a 0.22 pm syringe filter for in vivo animal experiments.
  • Human glioblastoma cell line U87MG cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured in DMEM containing high glucose (GIBCO, Carlsbad, CA), which was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were expanded in tissue culture dishes and kept in a humidified atmosphere of 5% C0 2 at 37°C. The medium was changed every other day. A confluent monolayer was detached with 0.05% Trypsin-EDTA, 0.01M PBS (pH 7.4) and dissociated into a single-cell suspension for further cell culture.
  • DMEM high glucose
  • FBS fetal bovine serum
  • penicillin-streptomycin penicillin-streptomycin
  • mice were anesthetized with isoflurane (5% for induction and 2% for maintenance in 100% 0 2 ) using a knock-down box.
  • isoflurane 5% for induction and 2% for maintenance in 100% 0 2
  • the biodistribution of 18 F-43 was evaluated in normal Sprague Dawley nude mice. Representative coronal microPET images at 0.5 and 3 h post injection of about 3.7 MBq (100 Ci) 18 F-43 were obtained. The activity was mainly accumulated in the liver and kidney. MicroPET quantification by measuring the ROI is shown in Figure 3B. The 10 kidney uptakes of 18 F-43 were 23.20 ⁇ 2.15 and 16.51 ⁇ 1.52 %ID/g at 30 min and 180 min post injection respectively. Low muscle uptakes were observed (2.98 ⁇ 0.66 and 1.59 ⁇ 0.57 %ID/g at 0.5 and 3 h post injection).
  • Diaminotetrazine 21 was re ared as follows.
  • Diisopropylethyl amine (0.032 ml, 0.195 mmol, 3 eq.) was added dropwise to the mixture. The reaction mixture was stirred for 24 hours at room temperature. The reaction was condensed in vacuo and then loaded onto a silica gel column using the minimum amount of MeOH required along with CH 2 CI 2 .

Abstract

La présente invention concerne un adduit de Diels-Alder d'un trans-cyclooctène avec une tétrazine, où l'adduit porte un substituant marqué avec un radionucléide. L'invention concerne un procédé de production d'une image par TEP ou autre image d'un organe chez un animal ou un humain consistant à former l'adduit de Diels-Alder chez l'animal ou l'humain. L'invention concerne des trans-cyclooctènes et des tétrazines appropriés pour la préparation des adduits.
PCT/US2011/044814 2010-07-23 2011-07-21 Ligature tétrazine-trans-cyclooctène pour la construction rapide de sondes marquées par un radionucléide WO2012012612A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US13/811,568 US10434197B2 (en) 2010-07-23 2011-07-21 Tetrazine-trans-cyclooctene ligation for the rapid construction of radionuclide labeled probes
CN201180041210.XA CN103221398B (zh) 2010-07-23 2011-07-21 用于快速构建放射性核素标记探针的四嗪-反式环辛烯连接反应
EP11810394.4A EP2595967B1 (fr) 2010-07-23 2011-07-21 Ligature tétrazine-trans-cyclooctène pour la construction rapide de sondes marquées par un radionucléide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US36717410P 2010-07-23 2010-07-23
US61/367,174 2010-07-23

Publications (2)

Publication Number Publication Date
WO2012012612A2 true WO2012012612A2 (fr) 2012-01-26
WO2012012612A3 WO2012012612A3 (fr) 2012-05-24

Family

ID=45497458

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/044814 WO2012012612A2 (fr) 2010-07-23 2011-07-21 Ligature tétrazine-trans-cyclooctène pour la construction rapide de sondes marquées par un radionucléide

Country Status (4)

Country Link
US (1) US10434197B2 (fr)
EP (1) EP2595967B1 (fr)
CN (1) CN103221398B (fr)
WO (1) WO2012012612A2 (fr)

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014081301A1 (fr) * 2012-11-22 2014-05-30 Tagworks Pharmaceuticals B.V. Activation de médicament bio-orthogonale
WO2014081303A1 (fr) * 2012-11-22 2014-05-30 Tagworks Pharmaceuticals B.V. Groupe clivable par voie chimique
WO2014117001A1 (fr) * 2013-01-25 2014-07-31 Memorial Sloan-Kettering Cancer Center Tétrazines/trans-cyclooctènes dans la synthèse en phase solide de peptides marqués
WO2014205126A1 (fr) 2013-06-19 2014-12-24 The Regents Of The University Of California Structures chimiques pour l'administration localisée d'agents thérapeutiques
WO2014182704A3 (fr) * 2013-05-06 2015-01-15 The General Hospital Corporation Sondes d'activation bio-orthogonales
EP3116486A4 (fr) * 2014-03-14 2017-11-08 Daniel J. Capon Immunoglobuline hybride contenant une liaison non peptidyle
US10130723B2 (en) 2014-03-14 2018-11-20 The Regents Of The University Of California TCO conjugates and methods for delivery of therapeutic agents
WO2018234488A1 (fr) 2017-06-23 2018-12-27 Basf Se Dérivés de cyclopropyle substitués
EP3389729A4 (fr) * 2015-12-15 2019-12-11 United Kingdom Research and Innovation Trans-cycloalcènes à contrainte conformationnelle pour radiomarquage
WO2020108720A1 (fr) 2018-11-30 2020-06-04 Københavns Universitet Nouveaux composés de tétrazine pour l'imagerie in vivo
US10828373B2 (en) 2015-09-10 2020-11-10 Tambo, Inc. Bioorthogonal compositions
WO2020239687A1 (fr) 2019-05-24 2020-12-03 Rigshospitalet Nouvelles sondes d'imagerie à base de tétrazines marquées au 18f, destinées au préciblage en imagerie de type tep
US11220556B2 (en) 2013-03-15 2022-01-11 Biomolecular Holdings Llc Hybrid immunoglobulin containing non-peptidyl linkage
US11253600B2 (en) 2017-04-07 2022-02-22 Tambo, Inc. Bioorthogonal compositions
EP4056546A1 (fr) 2021-03-11 2022-09-14 University of Copenhagen Radiomarquage 18f aliphatique d'un précurseur de tétrazine
WO2022189304A2 (fr) 2021-03-11 2022-09-15 University Of Copenhagen Radiomarquage au 18f aliphatique d'un précurseur de tétrazine
CN115974892A (zh) * 2022-12-27 2023-04-18 四川大学华西医院 三氮唑四嗪类化合物及其制备方法、应用
US11857636B2 (en) 2011-05-16 2024-01-02 Tagworks Pharmaceuticals B.V. Bio-orthogonal drug activation

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2522369A1 (fr) * 2011-05-09 2012-11-14 Koninklijke Philips Electronics N.V. Kit de pré-ciblage, procédé et agents utilisés avec celui-ci
US10556024B2 (en) 2013-11-13 2020-02-11 Whitehead Institute For Biomedical Research 18F labeling of proteins using sortases
AU2015236392A1 (en) * 2014-03-24 2016-10-20 Genia Technologies, Inc. Chemical methods for producing tagged nucleotides
WO2016025480A1 (fr) 2014-08-11 2016-02-18 The General Hospital Corporation Cyclo-octènes pour réactions bio-orthogonales
WO2017059397A1 (fr) 2015-10-01 2017-04-06 Whitehead Institute For Biomedical Research Marquage d'anticorps
EP3609885A1 (fr) * 2017-03-24 2020-02-19 Centre National De La Recherche Scientifique Plateforme tétrazine modulaire biocompatible
AU2018388467A1 (en) 2017-12-18 2020-06-11 Janssen Biotech, Inc. Radiolabeling of polypeptides
AU2019261451A1 (en) 2018-04-26 2020-12-03 Agenus Inc. Heat shock protein-binding peptide compositions and methods of use thereof
KR20220006613A (ko) 2019-05-10 2022-01-17 얀센 바이오테크 인코포레이티드 마크로사이클릭 킬레이터 및 이의 사용 방법
CN110954514B (zh) * 2019-12-02 2022-06-21 上海交通大学 适用于研究脂质体体内外分布的荧光示踪系统和方法
CN111138337B (zh) * 2019-12-26 2021-06-22 清华大学 用于生物正交反应的双环[4.1.0]庚烷亚硝基脲衍生物及其制备方法与应用
CN115667293A (zh) 2020-05-20 2023-01-31 詹森生物科技公司 使用转谷氨酰胺酶的糖基化单克隆抗体的位点特异性缀合
MX2023005462A (es) 2020-11-10 2023-05-22 Janssen Biotech Inc Compuestos macrociclicos y metodos para usarlos.
CN113248408B (zh) * 2021-04-30 2022-03-04 南京大学 一种多模态分子影像探针P-FFGd-TCO及其制备方法与应用
WO2022236011A1 (fr) 2021-05-07 2022-11-10 The Broad Institute, Inc. Transcriptomique à résolution spatio-temporelle avec résolution subcellulaire
WO2023084397A1 (fr) 2021-11-09 2023-05-19 Janssen Biotech, Inc. Composés macrocycliques et leurs utilisations diagnostiques
WO2023084396A1 (fr) 2021-11-09 2023-05-19 Janssen Biotech, Inc. Composés macrocycliques et leurs procédés de fabrication
CN114380813B (zh) * 2022-01-17 2023-06-06 厦门大学 一种vista靶向的探针及其制备方法和应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4053528A (en) 1973-12-28 1977-10-11 Hooker Chemicals & Plastics Corporation Process for the preparation of diels-alder adducts of halogenated cyclopentadienes
GB0428012D0 (en) * 2004-12-22 2005-01-26 Hammersmith Imanet Ltd Radiolabelling methods
US8236949B2 (en) * 2007-07-17 2012-08-07 University Of Delaware Tetrazine-based bio-orthogonal coupling reagents and methods
CN101723849B (zh) * 2008-10-10 2012-11-21 北京师范大学 18f标记氨基酸类衍生物、其制备方法及其制备报告肿瘤的正电子发射断层显像分子探针中的应用
WO2010051530A2 (fr) 2008-10-31 2010-05-06 The General Hospital Corporation Compositions et procédés d'administration d'une substance à une cible biologique

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
M. L. BLACKMAN; M. ROYZEN; J. M. FOX, J. AM. CHEM. SOC., vol. 130, 2008, pages 13518 - 13519
M. ROYZEN; G. P. A. YAP; J. M. FOX, J. AM. CHEM. SOC., vol. 130, 2008, pages 3760 - 3761
R. ROSSIN ET AL., ANGEW. CHEM. INT. ED., vol. 49, 2010, pages 3375 - 3378
S. GUHLKE ET AL., NUCL. MED. BIOL., vol. 21, no. 6, 1994, pages 819 - 825
See also references of EP2595967A4

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11857636B2 (en) 2011-05-16 2024-01-02 Tagworks Pharmaceuticals B.V. Bio-orthogonal drug activation
EP4245744A3 (fr) * 2012-11-22 2024-05-01 Tagworks Pharmaceuticals B.V. Groupe clivable par voie chimique
WO2014081303A1 (fr) * 2012-11-22 2014-05-30 Tagworks Pharmaceuticals B.V. Groupe clivable par voie chimique
US10927139B2 (en) 2012-11-22 2021-02-23 Tagworks Pharmaceuticals B.V. Chemically cleavable group
WO2014081301A1 (fr) * 2012-11-22 2014-05-30 Tagworks Pharmaceuticals B.V. Activation de médicament bio-orthogonale
WO2014117001A1 (fr) * 2013-01-25 2014-07-31 Memorial Sloan-Kettering Cancer Center Tétrazines/trans-cyclooctènes dans la synthèse en phase solide de peptides marqués
US11220556B2 (en) 2013-03-15 2022-01-11 Biomolecular Holdings Llc Hybrid immunoglobulin containing non-peptidyl linkage
WO2014182704A3 (fr) * 2013-05-06 2015-01-15 The General Hospital Corporation Sondes d'activation bio-orthogonales
US10517965B2 (en) 2013-05-06 2019-12-31 The General Hospital Corporation Bioorthogonal turn-on probes
US10130711B2 (en) 2013-06-19 2018-11-20 The Regents Of The University Of California Chemical structures for localized delivery of therapeutic agents
WO2014205126A1 (fr) 2013-06-19 2014-12-24 The Regents Of The University Of California Structures chimiques pour l'administration localisée d'agents thérapeutiques
US10953098B2 (en) 2013-06-19 2021-03-23 The Regents Of The University Of California Chemical structures for localized delivery of therapeutic agents
US10130723B2 (en) 2014-03-14 2018-11-20 The Regents Of The University Of California TCO conjugates and methods for delivery of therapeutic agents
US10342882B2 (en) 2014-03-14 2019-07-09 The Regents Of The University Of California TCO conjugates and methods for delivery of therapeutic agents
EP4169944A1 (fr) * 2014-03-14 2023-04-26 Biomolecular Holdings LLC Procédé de préparation d'immunoglobuline hybride contenant une liaison non peptidyle
US10806807B2 (en) 2014-03-14 2020-10-20 The Regents Of The University Of California TCO conjugates and methods for delivery of therapeutic agents
EP3116486A4 (fr) * 2014-03-14 2017-11-08 Daniel J. Capon Immunoglobuline hybride contenant une liaison non peptidyle
US11066459B2 (en) 2014-03-14 2021-07-20 Biomolecular Holdings Llc Hybrid immunoglobulin containing non-peptidyl linkage
US10828373B2 (en) 2015-09-10 2020-11-10 Tambo, Inc. Bioorthogonal compositions
EP3389729A4 (fr) * 2015-12-15 2019-12-11 United Kingdom Research and Innovation Trans-cycloalcènes à contrainte conformationnelle pour radiomarquage
US11253600B2 (en) 2017-04-07 2022-02-22 Tambo, Inc. Bioorthogonal compositions
WO2018234488A1 (fr) 2017-06-23 2018-12-27 Basf Se Dérivés de cyclopropyle substitués
WO2020108720A1 (fr) 2018-11-30 2020-06-04 Københavns Universitet Nouveaux composés de tétrazine pour l'imagerie in vivo
WO2020239687A1 (fr) 2019-05-24 2020-12-03 Rigshospitalet Nouvelles sondes d'imagerie à base de tétrazines marquées au 18f, destinées au préciblage en imagerie de type tep
EP4056546A1 (fr) 2021-03-11 2022-09-14 University of Copenhagen Radiomarquage 18f aliphatique d'un précurseur de tétrazine
WO2022189304A2 (fr) 2021-03-11 2022-09-15 University Of Copenhagen Radiomarquage au 18f aliphatique d'un précurseur de tétrazine
CN115974892A (zh) * 2022-12-27 2023-04-18 四川大学华西医院 三氮唑四嗪类化合物及其制备方法、应用
CN115974892B (zh) * 2022-12-27 2023-09-08 四川大学华西医院 三氮唑四嗪类化合物及其制备方法、应用

Also Published As

Publication number Publication date
US20130266512A1 (en) 2013-10-10
EP2595967A2 (fr) 2013-05-29
CN103221398B (zh) 2016-03-23
WO2012012612A3 (fr) 2012-05-24
CN103221398A (zh) 2013-07-24
EP2595967A4 (fr) 2013-12-04
US10434197B2 (en) 2019-10-08
EP2595967B1 (fr) 2016-03-23

Similar Documents

Publication Publication Date Title
US10434197B2 (en) Tetrazine-trans-cyclooctene ligation for the rapid construction of radionuclide labeled probes
Boros et al. RGD conjugates of the H2dedpa scaffold: synthesis, labeling and imaging with 68Ga
Decristoforo et al. 68 Ga-and 111 In-labelled DOTA-RGD peptides for imaging of αvβ3 integrin expression
Price et al. H 6 phospa-trastuzumab: bifunctional methylenephosphonate-based chelator with 89 Zr, 111 In and 177 Lu
ES2547712T3 (es) Métodos y composiciones para el marcado de proteínas
Läppchen et al. DOTA-tetrazine probes with modified linkers for tumor pretargeting
Gasser et al. Synthesis, copper (II) complexation, 64Cu-labeling, and bioconjugation of a new bis (2-pyridylmethyl) derivative of 1, 4, 7-triazacyclononane
AU2021202665A1 (en) Radio-pharmaceutical complexes
Zhou et al. An efficient method for labeling single domain antibody fragments with 18F using tetrazine-trans-cyclooctene ligation and a renal brush border enzyme-cleavable linker
US20200268913A1 (en) Radioactive drug
US20130343990A1 (en) Radiolabelled octreotate analogues as pet tracers
Liu et al. A new 18 F-heteroaryltrifluoroborate radio-prosthetic with greatly enhanced stability that is labelled by 18 F–19 F-isotope exchange in good yield at high specific activity
Gotzmann et al. Synthesis, 64 Cu-labeling and PET imaging of 1, 4, 7-triazacyclononane derived chelators with pendant azaheterocyclic arms
Al Jammaz et al. Novel synthesis and preclinical evaluation of folic acid derivatives labeled with 18F-[FDG] for PET imaging of folate receptor-positive tumors
Al Jammaz et al. Rapid synthesis and in vitro and in vivo evaluation of folic acid derivatives labeled with fluorine-18 for PET imaging of folate receptor-positive tumors
Fernández et al. Synthesis, in vitro and in vivo characterization of two novel 68Ga-labelled 5-nitroimidazole derivatives as potential agents for imaging hypoxia
Choi et al. N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: Influence of isomeric substitution on radiolabeling and target cell residualization
Wharton et al. H4picoopa─ Robust Chelate for 225Ac/111In Theranostics
Vaidyanathan et al. SIB-DOTA: A trifunctional prosthetic group potentially amenable for multi-modal labeling that enhances tumor uptake of internalizing monoclonal antibodies
Singh et al. A homodimeric bivalent radioligand derived from 1-(2-methoxyphenyl) piperazine with high affinity for in vivo 5-HT1A receptor imaging
EP3554559B1 (fr) Procédé de marquage des biomolécules de ciblage sensibles et thermosensibles avec des composés à base de technétium
EP4043041A1 (fr) Cxcr4-ligands pour usage diagnostique et thérapeutique et leurs précurseurs
US20200188540A1 (en) Conformationally strained trans-cycloalkenes for radiolabeling
Yang et al. Synthesis and evaluation of 111 In-labeled d-glucose as a potential SPECT imaging agent
US20240051976A1 (en) A Tracer Compound and a Preparation Method Thereof

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201180041210.X

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11810394

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2011810394

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 13811568

Country of ref document: US